Virus-derived siRNA mediated inhibition of PRRSV replication in pigs [PAM]

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). It clinically manifests as fever, bleeding, and respiratory symptoms in pigs of different age groups, as well as reproductive disorders, causing huge economic losses to the global swine industry. RNA interference (RNAi), a highly conserved mechanism mediating post-transcriptional gene silencing, exerts antiviral effects by cleaving viral double-stranded RNA to generate virus-derived siRNA (vsiRNA). VsiRNA-mediated antiviral immunity is widespread in plants and invertebrates, while in mammals, studies have focused on reducing PRRSV viral load in pigs via siRNA delivery. Therefore, PRRSV strains (LY and LY-R) were primarily selected as virus models to infect PAM and Marc-145 cells, respectively. Then, miRNA and vsiRNA analysis were performed and bioinformatics analysis revealed multiple differentially expressed miRNAs following PRRSV infection. Further enrichment analysis indicated that these miRNAs were mainly enriched in metabolic, endocytic, and phosphorylation signaling pathways. Additionally, in Marc-145 cells infected with PRRSV-LY, high abundance of vsiRNA was detected within the PRRSV-N genome. The N-vsiRNA was inserted into vector psilencer4.1. to construct a PS-N plasmid, which is capable of exerting anti-PRRSV (LY, LY-R, and NADC30-like) activity in Marc-145 cells. Finally, the PS-N plasmid was formulated with PLGA-PEI to prepare a nanoparticle mixture, which was delivered to pigs via intramuscular injection to alleviate tissue damage caused by PRRSV-LY infection to a certain extent. In summary, the porcine RNAi system and vsiRNA generated via viral cleavage play a critical role in inhibiting virus replication. This further demonstrates the feasibility of vsiRNA-based antiviral strategies and offers a reference for the prevention and control of various RNA viruses. Overall design: For PAM experiments, the infection level of PRRSV LY and LY-R is 0.1 MOI. They were incubated at 37? with 5% CO2 for 2 h and then cleaned using RPMI 1640. The non-infected virus cells for the Blank group. The RNA samples were collected according to the indicated time (24 h). The group contain the Blank, HPPRRSV, and PRRSV.